PROJECT SUMMARY The family of Filoviridae consisting of five ebolaviruses and two marburgviruses are causative agents of severe hemorrhagic fever with human case fatality rates of 50-90%. The 2014-2015 epidemic of Ebola virus disease in West Africa was unprecedented both in geographic scope and size and highlighted the extent of the threat filoviruses could pose to global public health and the urgent need for development of therapeutics. Among the five ebolavirus species, the Zaire and Sudan subtypes (EBOV and SUDV, respectively) are the most pathogenic and both have resulted in recurring outbreaks. Furthermore, Bundibugyo virus (BDBV) has also caused two sizable outbreaks over the past decade. However, current filovirus antibody therapies are narrowly specific to Zaire Ebola virus and ineffective against SUDV and BDBV. The goal of this proposal is to develop a bispecific antibody (Bis-mAb) therapeutic with broad protection against all ebolaviruses that have caused human disease. In the Phase I of this STTR we created a prototype Bis-mAb that combines the antigen binding variable domains of a non-neutralizing pan-ebolavirus antibody with an antibody against a cryptic epitope within the receptor binding site of ebolavirus glycoprotein that becomes exposed only within the endosomes. The Bis- mAb colocalizes to the endosomes with the virus where the antibody against the cryptic epitope becomes effective due to proteolytic cleavage of the glycoprotein by cysteine cathepsins. This approach enabled us to generate a ?Trojan Horse? that co-opts the virus to deliver a lethal weapon into the endosomes leading to effective virus neutralization. This prototypic pan-ebolavirus Bis-mAb (FVM09~MR72) potently neutralized all five ebolavirus species while a cocktail of the parental IgGs was ineffective. In mice, FVM09~MR72 protected against lethal challenge with EBOV and SUDV, the most divergent ebolaviruses. In the Phase II project, we propose to demonstrate protective efficacy of the product in nonhuman primates and ferrets to achieve a critical milestone that would justify clinical development. In Aim 1 we will produce the Bis-mAb in CHO cells by transient transfection at scale required for large animal studies. Stable cell lines will be also generated for use in future manufacturing of the product. In Aim 2, we will evaluate the pharmacokinetics of the Bis-mAb and conduct proof of principal efficacy against SUDV and EBOV in NHPs and against BDBV in ferrets. This work represents a partnership between academic (Einstein) and commercial (Integrated BioTherapeutics) entities to develop a cross-neutralizing therapeutic Bis-mAb. The approach leverages proprietary antibodies, technology platforms, and complementary expertise from both partners.